Long Non-coding RNA Expression Profile And Functional Analysis In Children With Fulminant Myocarditis

BackgroundMyocarditis is an inflammatory process caused by diverse etiologies with diffuse or focal myocardial interstitial inflammatory cells infiltration and myocardium necrosis or degeneration.Its clinical manifestation ranges from unobrious symptoms in mild patients to severe symptoms with heart failure,cardiac shock,or even death.Some patients could develop dilated cardiomyopathy.Acute fulminant myocarditis（AFM）is a type of myocarditis that can arise quickly,progress rapidly and lead to severe alteration in hemodynamics,damaging heart failuler,and even cardiogenic shock.The pathogenesis mechanism mainly includes direct virus damage and immune damage.However,the molecular mechanism of AFM has not been demonstrated.Long non-coding RNA（lncRNA）are defined as transcripts of more than 200 nucleotides that are not translated into proteins.LncRNA participates in various developmental processes in transcriptional,post-transcriptional,protein translational,protein post-translational,or epigenetic regulation.Recent years,an increasing number of lncRNAs are emerging as having roles in arrhythmia,heart failure and atherosclerosis.However,data related to myocarditis and lncRNA are scarce.ObjectiveTo determine the differential expression profiles of IncRNA and mRNA in peripheral leukocytes with acute fulminant myocarditis and healthy children,and then select lncRNA related to AFM to find novelly potential biomarker and providing a new clue for follow-up basic reserch.MethodIn this study,we recruit 15 test group and 15 control group.Among them 3 test group with typical clinical symptoms and 3 control group were used to generate biotinylated cDNA targets with the Sino Human ceRNA array V3.0（Shanghai Sinomics Corporation,Shanghai,China）.Then we use bioinformatics anlysis including differential expression analysis of lncRNA and mRNA,GO and KEGG pathway anlysis,IncRNA-miRNA interaction network,cis/trans IncRNA target prediction and co-expression network anlysis.Nest,5 test group and 5 control group were test by qRT-PCR to verify the microarray data,including 3 test group and 3 control group detected by microarray.Finally,15 test groups and 15 control groups,including the above 5 test groups and 5 control groups,used qRT-PCR to verify the expression of NONHSAT253897.1.Results1.In total,using a 2/0.5-fold change and P<0.05 as the cutoff criteria,3101 IncRNAs displayed differential expression in AFM patients,including 1645 upregulated IncRNAs and 1456 downregulated lncRNAs.In addition,a total of 2170 mRNAs were dysregulated;among them,733 were upregulated and 1437 were downregulated.2.The most prominent GO terms were pyramidal neuron development and differentiation,negative regulation of complement activation,positive regulation of transcription from RNA polymerase Ⅲ promoter,T-helper 17 cell differentiation,typeⅠ interferon biosynthetic process,negative regulation of multicellular organism growth,thyroid hormone metabolic process,MHC class Ⅱ biosynthetic process,positive regulation of antigen receptor-mediated signaling pathway,T-helper 2 cell differentiation,forebrain neuron development,T-helper cell differentiation,negative regulation of interleukin-6 production,positive regulation of organ growth,regulation of tolerance induction,regulation of B cell apoptotic process,regulation of T cell differentiation in thymus,interferon-alpha production,T-helper 1 type immune response.3.The notable pathways in KEGG pathway analysis include hematopoietic cell lineage,T cell receptor signaling pathway,complement and coagulation cascades,Notch signaling pathway,antigen processing and presentation,ErbB signaling pathway,Toll-like receptor signaling pathway,natural killer cell mediated cytotoxicity,Jak-STAT signaling pathway,Fc epsilon RI signaling pathway,calcium signaling pathway,Leukocyte transendothelial migration,MAPK signaling pathway,Fc gamma R-mediated phagocytosis.4.Cis/trans IncRNA target prediction was performed for differentially expressed IncRNAs to investigate whether they can regulate genes and to determine the signaling pathways associated with AFM.The results show that IncRNA is mainly involved in transcription regulation by transcription factors in T cell receptor signaling pathway and Jak-STAT signaling pathway.5.LncRNA-mRNA coexpression network of T cell receptor and Jak-SATA signaling pathways was constructed to predict gene function.It was shown that mRNA IL21R、IL23R、AKT3、CD3E、STAT4、LAT、CCND2、CD247、NFATC2、LCK、ITK、CDK4、CD8B、CD3D、PLCG、IL12RB1、IL10RB、IL10、IL4R、SOS2 play a vital role in AFM.6.LncRNA-microRNA interaction network predict that hsa-miR-3653-5P act as sponges by binding NONHSAT254241.1 or NONHSAT150563.1 to regulate CDK4.In addition,has-miR-370-3P act as sponges by binding NR₀31607.1 or NONHSAT197107.1 to regulate IL-10.7.qRT-PCR analysis validate that the results of NONHSAT253897.1,NONHSAT177112.1,NONHSAT256669.1,NR₁26169.1,NONHSAT232454.1,IL10 and SOS2 are consistent with the microarray data.However,the results for NONHSAT234238.1 are inconsistent with the trend sown by the microarray data.Conclusions1.LncRNA and mRNA play an important role in the occurrence and development of AFM.2.NONHSAT253897.1 may be a potential biomarker in children with AFM.